Edited by: Vincent Kam Wai Wong, Macau University of Science and Technology, Macau
Reviewed by: Jianming Guo, Nanjing University of Chinese Medicine, China; Aihua Zhang, Heilongjiang University of Chinese Medicine, China
*Correspondence: Wen-Bo Wang,
This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology
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Postmenopausal osteoporosis (PMOP) is a common metabolic bone disease in postmenopausal women in the Worldwide, and seriously affects the quality of life of middle-aged and elderly women. Therefore, there is an urgent need to discover a highly effective drug for PMOP treatment. In this study, ultra-high performance liquid tandem quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to analyze the urine metabolic profiling and potential biomarkers, the relevant metabolic network of PMOP rats, and further to evaluate the intervention effect of Eleutheroside E (EE) against PMOP. Using multivariate statistical analysis combined with UPLC-Q/TOF-MS, a total of 27 biomarkers were identified, which related with 16 metabolic pathways, mainly involving steroidogenesis, beta oxidation of very long chain fatty acids, glutathione metabolism, carnitine synthesis, estrone metabolism, oxidation of branched chain fatty acids, etc. After treatment of EE, these biomarkers were markedly regulated, mainly involving steroid hormone biosynthesis, arachidonic acid metabolism, primary bile acid biosynthesis, indicating that EE had the therapeutic effect on PMOP. This study identified the potential urine metabolic markers and related metabolic pathways of the PMOP, explained the metabolic effect and pharmacological mechanisms of EE against PMOP, and provided a basis for the pharmacological study of EE.
Osteoporosis (OP) is a systemic skeletal disease, which is characterized by a decrease in bone mass and destruction of bone tissue microstructure, leading to an increased risk of fracture (
A previous study showed that the Acanthophanax senticosus has efficacy on the ovariectomized osteoporosis model rats, could affect the endogenous metabolites related metabolic mechanism, and has provided a therapeutic basis for the PMOP treatment. Eleutheroside E (EE) is an important active ingredient from Acanthopanax senticosus, however, its therapeutic mechanism for PMOP is unclear. Metabolomics method was used to explore the potential biomarkers and therapeutic targets for natural products. Therefore, this study was to explore and evaluate the therapeutic targets of EE on the ovariectomized osteoporosis model rats by visually observe the dynamic changes of the urine metabolic profiling, and to find biomarkers and related metabolic pathways for PMOP, in order to provide experimental basis for the development of new anti-osteoporosis drugs.
Acetonitrile and methanol was purchased from Merck Drugs & Biotechnology (HPLC grade, Germany); formic acid was obtained from Kemiou Chemical Reagent Co. Ltd. (HPLC grade, Tianjin, China); ultrapure water was provided by Watson’s Food & Beverage Co., Ltd (Guangzhou, China). Nylestriol was provided from Vikchi Biotech Co., Ltd. (Sichuan, China); normal saline was provided by Harbin Medisan Pharmaceutical Co., Ltd. (Harbin, China). Chloral hydrate obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). Sodium penicillin for injection was purchased from Harbin pharmaceutical Group Holding Co., Ltd. (Harbin, China). All other reagents are of analytical grade. The standard chemicals of EE with 99.5% was purchased from Chengdu Biopurity Phytochemicals Ltd., (Sichuan, China); and the chromatogram of EE was shown in
Wistar rats (female, weight 220–240g) were provided from the Experimental Animal Center of Harbin Medical University, and randomly divided into sham operation group (Sham, n=8), model group (M, n=8), the EE group (n=8), and positive control group (PC, n=8). After adapting to the environment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (0.3g·kg-1). Except for Sham, bilateral ovaries were removed by surgical emasculation in the remaining five groups to prepare PMOP rat model (
Urine in each group was collected for 12 h at night every 4 days for a total of 12 weeks. Fresh urine samples were initially processed by centrifugation (4°C, 10 min at 10,000 rpm), and the supernatant was frozen at -80°Cfor later using. Before analysis, urine samples were thawed at room temperature. After vortexing for 10 s, the samples were centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was passed through a 0.22 μm filter and transferred to a sample cup for UPLC-MS analysis.
An ultra LC 100 system (AB. Ltd., USA) equipped with an ACQUITY UPLC™ T3 Column (100 mm×2.1 mm i.d., 1.8 μm, Waters Corp., USA) was used to analyze urine samples. The column temperature was maintained at 45°C. The 4 μl was set as the injection volume. And all samples had been kept at 4°C sample room during the data collection period. The mass spectrometry (MS) was performed usinga Triple TOFR 5600+ high-definition quadrupole time-of-flight mass spectrometry (AB, USA) with electrospray ion (ESI) source. The full-scan mass number ranges from 50 to 1,000, and the eight strongest fragment ions over 100 CPS are scanned for the daughter ions. Dynamic background deduction was enabled, and the automatic correction system was automatically adjusted and corrected MS and MS/MS.
The urine metabolic profiling data by UPLC-Q/TOF-MS was imported into ProgenesisR QI software (Waters, USA) and Metaboanalyst for peak alignment and standardization. The three-dimensional data of ion retention time– mass to charge ratio-peak intensity of metabolites were extracted. Metaboanalyst was used for multivariate statistical analysis. The biomarkers of PMOP were identified by searching various metabolomics databases such as Human Metabolome Database (HMDB), Kyoto Encyclopedia of Genes and Genomes (KEGG), and combining MS/MS fragmentation information. The identified compounds mass error range was ±5ppm. And then MetaboAnalyst 4.0 was performed for metabolic pathway analysis and visualization.
The previous studies displayed that PMOP rat model was replicated by the ovariectomy (
The principal component analysis (PCA) scores plot of urine samples from the control group and the model group. **
In order to search the potential endogenous metabolites that played the key role in clustering, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the urine metabolic profiling data of rats in Sham and M groups to obtain the Scores plot. Ions with VIP-value greater than 1.0 and t-test results between groups (P-value) less than 0.05 were selected as potential biomarkers. Endogenous metabolites were screened by HMDB and KEGG databases, combined with MS/MS fragment information of compounds to determine the chemical structure of potential biomarkers. Finally, a total of 27 biomarkers were identified in PMOP model, including 15 in positive ion mode, 12 in negative ion mode. The details of identified biomarkers were shown in the
The heatmap analysis for urine potential biomarkers in the control group and the model group.
The above identified of 27 PMOP model biomarkers were imported into the Pathway Analysis module of MetaboAnalyst to analyze the metabolic network systematically. A total of 10 related metabolic pathways were acquired, including steroidogenesis, beta oxidation of very long chain fatty acids, glutathione metabolism, carnitine synthesis, estrone metabolism, oxidation of branched chain fatty acids, mitochondrial beta-oxidation of short chain saturated fatty acids, bile acid biosynthesis, mitochondrial beta-oxidation of long chain saturated fatty acids, androgen and estrogen metabolism, fatty acid metabolism, arginine and proline metabolism, glycine and serine metabolism, tryptophan metabolism, arachidonic acid metabolism, and tyrosine metabolism (
The metabolite sets enrichment overview of urine biomarkers in t postmenopausal osteoporosis.
The network pathways analysis of urine biomarkers in postmenopausal osteoporosis by the MetaboAnalyst.
Multivariate statistical analysis was performed on the urinary metabolic profiling data of rats in Sham, M, EE, and PC group on the 28th day. From the PCA scores plot (
The principal component analysis (PCA) score plot of all the groups in urine metabolism profile by eleutheroside E against postmenopausal osteoporosis.
The principal component analysis (PCA) 3-D scores plot of urine metabolism profile samples from eleutheroside E against postmenopausal osteoporosis.
The heatmap analysis for urine potential biomarkers in eleutheroside E against postmenopausal osteoporosis.
The metabolic pathways regulation analysis of eleutheroside E against postmenopausal osteoporosis.
In this study, we established a UPLC-Q/TOF-MS metabolomics method to analyze urine samples of PMOP model rats, and observed the endogenous metabolites changes in the urine of rat models. From the perspective of metabolomics, the normal metabolic network of rats was disturbed after model replication, and the metabolic process in PMOP rat model was also demonstrated. It can be seen that the metabolic profiling of the model rats had a large change on the 28th day after model replication. Then the established analysis method was used to analyze the urine metabolic profiling of M group and Sham group on the 28th day of model replication. The two groups were clearly separated, which indicates that the PMOP model was successfully replicated from the metabolomics level. Next, using multivariate statistical analysis combined with multiple metabolomics databases and matching MS/MS fragmentation information, 27 potential biomarkers related to PMOP were found, resulting in disturbances of 10 metabolic pathways, mainly involving lipid metabolism and amino acid metabolism.
The disturbance of lipid metabolism was dominant, includingsteroid hormone biosynthesis, arachidonic acid metabolism, and primary bile acid biosynthesis. Research suggests that there are many factors play a role in the formation of the PMOP, including metabolic disorders, especially hormonal imbalances (
Prostaglandin G2 (PGG2) is the main product synthesized from arachidonic acid by cyclooxygenase (COX) and can be further metabolized to prostaglandin E2 (PGE2) (
Cholesterol is an indispensable and important substance for animal tissue cells, and is the raw material for the synthesis of bile acids and steroid hormones. It can bind to estrogen receptor-associated receptor alpha topromote osteoclast formation, survival, and cell fusion (
The formation of PMOP is accompanied by significant disturbances in lipid metabolism, as well as significant disturbances in amino acid metabolism. Amino acid metabolism plays an important role in bone metabolism (
Arginine, proline, leucine, and phenylalanine can enhance the effect of insulin-like growth factor 1 (
Pyroglutamic acid is produced byglutathione (GSH) under the action of CHAC, it can produce
Ovariectomized rats were administrated of EE for 28 consecutive days, 21 metabolites were call backed, among which 11 metabolites were significantly regulated, namely pyroglutamic acid, N2,N2-dimethylguanosine, dopaquinone, 5-acetamidovalerate, taurocholic acid, cholesterol sulfate, 17-hydroxyprogesterone, cholic acid, cholic acid glucuronide, 7a,12a-dihydroxy-3-oxo-4-cholenoic acid, 21-deoxycortisol. It mainly involves steroid hormone biosynthesis, primary bile acid biosynthesis, glutathione metabolism, and tyrosine metabolism. These results show that EE might inhibit cholesterol to synthesize bile acid, enhance the selective binding ability of EE to estrogen receptor, and promote the function of adrenaline, and then alleviate the dysfunction of HPA axis and increase the secretion of glucocorticoid; at the same time, EE might also reduce cell damage by adjusting the oxidative stress state in the body. Under the action of insulin and EGF regulatory factors, the bone density and bone strength of the ovariectomized rats were increased, and bone reconstruction was promoted, thus delaying the PMOP process. Combined with the previous results of blood metabolomics (
In this study, with the multivariate statistical analysis and UPLC-Q/TOF-MS, a total of 27 biomarkers were identified, which related with 16 metabolic pathways, mainly involving steroidogenesis, beta oxidation of very long chain fatty acids, glutathione metabolism, carnitine synthesis, estrone metabolism, oxidation of branched chain fatty acids, etc. These biomarkers were markedly regulated by EE, mainly involving steroid hormone biosynthesis, arachidonic acid metabolism, primary bile acid biosynthesis, indicating that EE had the therapeutic effect on PMOP. This study identified the potential urine metabolic markers and related metabolic pathways of the PMOP, explained the metabolic effect and pharmacological mechanisms of EE against PMOP, and provided a basis for the pharmacological study of EE.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
The animal study was reviewed and approved by First Affiliated Hospital of Harbin Medical University.
W-BW conceived and designed the experiments. Y-SM, Z-JH, YL, B-BZ, and J-MW performed the experiment and analyzed the data. Y-SM wrote the paper. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: